Peptide hormones and neurotransmitters are usually produced as larger pro-peptides, requiring a series of enzymes to generate the bioactive peptide (Docherty, K. and Steiner, D. F. (1982) Ann. Rev. Physiol., 44, 625-638; Eipper, et al. (1986) Trends. Neurosci., 9, 463-468; Steiner, D. F. (1991). In: Peptide Biosynthesis and Processing, Fricker, L. D. (ed.), pp. 1-16, CRC Press, Boca Raton). Most of these cleavages occur at specific base residue sites, and enzymes that initially cleave the precursor have been identified (Smeekens, S. P. and Steiner, D. F. (1990) J. Biol. Chem. 265, 2997-3000; Seidah, et al. (1990) DNA Cell. Biol., 9, 415-424; Hosaka, et al. (1991) J. Biol. Chem., 266, 12127-12130; Kiefer, et al. (1991) DNA Cell Biol., 10, 757-769; Nakagawa, et al. (1993) J. Biochem., 113, 132-135). Following this initial cleavage, a carboxypeptidase is then usually required to remove the C-terminal basic residues from the peptide to produce the bioactive moiety (Fricker, L. D. (1988) Ann. Rev. Physiol., 50, 309-321; Fricker, L. D. (1991). In: Peptide Biosynthesis and Processing, Fricker, L. D. (ed.), pp. 199-230, CRC Press, Boca Raton). For many years, a single carboxypeptidase was thought to be involved with the processing of most secreted peptides (Fricker, L. D. (1988) Ann. Rev. Physiol., 50, 309-321; Fricker, L. D. (1991). In: Peptide Biosynthesis and Processing, Fricker, L. D. (ed.), pp. 199-230, CRC Press, Boca Raton). This enzyme is alternatively known as carboxypeptidase E (CPE), carboxypeptidase H, and ankephalin convertase, and has been designated EC 3.4.17.10 (Webb, E. C. (1986) Eur. J. Biochem., 157, 1-26). CPE was initially discovered to be associated with the production of enkephalin in the adrenal medulla (Fricker, L. D. and Snyder, S. H. (1982) Proc. Natl. Acad. Sci. USA 79, 3886-3890), and has been found in all neuroendocrine tissues (Fricker, L. D. (1988) Ann. Rev. Physiol., 50, 309-321; Fricker, L. D. (1991). In: Peptide Biosynthesis and Processing, Fricker, L. D. (ed.), pp. 199-230, CRC Press, Boca Raton; Schafer, et al. (1994) J. Neurosci., 13, 1258-1279; Zheng, et al. (1994) J. Neurosci., 14, 4656-4673). However, the view that CPE is the only intracellular peptide-processing carboxypeptidase has been challenged by the finding that mice with the fat mutation are still capable of producing insulin, albeit at lower levels. (Naggert, et al. (1995) Nature Genetics, 10, 135-142).
The fat mutation has been mapped to the CPE locus on Chromosome 8, and a point mutation has been found in the coding region (Naggert, et al. (1995) Nature Genetics, 10, 135-142). The point mutation converts Ser.sup.202 into a Pro residue. When this mutation is created in the homologous rat CPE and the protein expressed in Sf9 cells using the Baculovirus system, the enzyme is inactive and is not secreted into the medium (Naggert, et al. (1995) Nature Genetics, 10, 135-142). Comparison of the enzyme activity measured between mutant and wild type CPE in the Baculovirus system showed that the mutation resulted in less than 0.1% activity of the wild type CPE. However, the levels of CPE-like-activity in Cpe.sup.fat /Cpe.sup.fat mouse pituitary and pancreatic islets were found to be 5-10% of the levels in tissues from control mice (Naggert, et al. (1995) Nature Genetics, 10, 135-142). Furthermore, the C-terminal processing of insulin is not completely eliminated in the Cpe.sup.fat /Cpe.sup.fat (Naggert, et al. (1995) Nature Genetics, 10, 135-142), suggesting that an active carboxypeptidase is present in the insulin secretory pathway of the Cpe.sup.fat /Cpe.sup.fat mouse.
A newly reported enzyme, carboxypeptidase D (CPD), may be involved in the processing of secretory pathway peptides and partially compensate for the defective CPE in Cpe.sup.fat /Cpe.sup.fat mice (Song, L. and Fricker, L. D. (1995) J. Biol. Chem., 270, 25007-25013). CPD is present in bovine pituitary and adrenal (Song, L. and Fricker, L. D. (1995) J. Biol. Chem., 270, 25007-25013) and in many rat tissues (L. Song and L. Fricker, unpublished). In contrast, CPE is present mainly in neuroendocrine tissues, and undetectable levels in liver (Zheng, et al. (1994) J. Neurosci., 14, 4656-4673; Fricker, et al. (1989) Mol. Endocrinol., 3, 666-673). The major difference between the two enzymes is the size; CPE is approximately 50-56 kDa, whereas CPD is 180 kDa in bovine pituitary (Song, L. and Fricker, L. D. (1995) J. Biol. Chem., 270, 25007-25013) and 100-180 kDa in various rat tissues (L. Song and L. Fricker, in preparation). CPD is not recognized by antisera raised against the N- or C-terminal regions of CPE (Song, L. and Fricker, L. D. (1995) J. Biol. Chem., 270, 25007-25013).
It has recently been found that a mutation responsible for obesity in the Cpe.sup.fat /Cpe.sup.fat mouse maps to the CPE gene locus, and that a point mutation is found within the Cpe.sup.fat /Cpe.sup.fat mouse gene (Naggart, et al. Nature Genetics 10, 135 (1995)). This point mutation causes CPE to be inactive, and degrades within the cell (Varlamov, et al. J. Biol. Chem., 271, 13981 (1996)). As stated above, the absence of CPE leads to defects in the processing of insulin (Naggart, et al. Nature Genetics 10, 135 (1995)). In the brain, a defect was found in the processing of enkephalin (Fricker, et al. J. Biol. Chem., 271:30614-30624 (1996)), and another group found a defect in the processing of neurotensin and melanin-concentrating hormone (Rovere, et al., Endocrinology, 137, 2954 (1996)). In all cases, the amount of the correctly processed peptide is decreased in the Cpe.sup.fat /Cpe.sup.fat mouse. Whereas in normal mouse tissues there are virtually no peptide intermediates containing C-terminal basic residues, these intermediates accumulate to high levels in the Cpe.sup.fat /Cpe.sup.fat mouse.
It is likely that the obesity-causing defect in the Cpe.sup.fat /Cpe.sup.fat mouse is due to a novel peptide because the physiological changes of these mice do not resemble those produced by the known peptides. The Cpe.sup.fat /Cpe.sup.fat mouse is not extremely hyperphagic, and regulate body weight more through metabolic changes than through increased feeding. Peptides such as neuropeptide Y are not likely candidates because they stimulate feeding, and the absence of these peptides would be expected to produce skinny mice. Although there are peptides known to inhibit feeding, the primary change in the Cpe.sup.fat /Cpe.sup.fat mouse is metabolic, and is not likely to be mediated by the known peptides. Accordingly, a method for the identification of peptides that are not correctly processed in the Cpe.sup.fat /Cpe.sup.fat mouse would be extremely useful, as peptides identified by this method have the potential to be used in therapeutics to control obesity.
Cells communicate with each other using a variety of signaling molecules. Many of these signaling molecules are peptides that are secreted from one cell type, and then bind to a receptor on a second cell type. A large number of therapeutics are based on peptides. In some cases, the peptide itself is the therapeutic (for example, insulin). In other cases, the actual therapeutic is a compound which mimics the action of the peptide by binding to the peptide's receptor and either stimulating or blocking the receptor action (for example, morphine, which binds to the receptors for enkephalin and other endogenous opiate peptides).
Current approaches for identifying individual substrates for enzymes and receptors are extremely time consuming. Over the past 50 years a number of peptides have been discovered largely by chance, and not through a systematic search based on the relative abundance of each peptide in the brain or other tissues. For example, the enkephalins were discovered by their ability to mimic the action of an opiate drug in a bioassay. If plants did not produce opiates, then it is unlikely that the enkephalin would have been discovered. It is widely estimated that only half of all peptides have been discovered. A rapid method of identifying peptides and other molecules that interact with enzymes and receptors is greatly needed for identifying all enzyme and receptor substrates. For example, many G-protein-coupled receptors bind peptides, and it is likely that a large number of these orphan receptors have undiscovered peptide ligands. Specifically, polypeptide hormones, after binding to their membrane-located receptors, induce changes in the membrane-bound G proteins, which are currently little understood. A rapid method of identifying the polypeptide hormones that bind to the membrane-located receptors would aid tremendously in understanding membrane-bound G-proteins. A rapid method of identification of enzyme substrates would decrease the time required for identifying possible drugs, and would accordingly decrease the overall time and expenses required for the process of the research, design and development of drugs.